Genome Editing of the Murine Calreticulin Gene Using the CRISPR/Cas9 and CPF1 System

<Background> Abnormalities of the Calreticulin (CALR) gene are frequently observed in human Myeloproliferative Neoplasms (MPN). Small deletions and insertions in the last exon of CALR, leading to frame-shift mutations, are common genetic alterations in this disease. Genome editing by the CRISPR/Cas9 (Clustered Regularly-Interspaced Short Palindromic Repeats and CRISPR-Associated proteins 9) system is one of ways to manipulate genes in normal/abnormal mammalian cells. Recently, CPF1 (CRISPR from Prevotella and Francisella 1) which is a novel single-component RNA-guided endonuclease has been identified. CPF1 cleaves double-strand DNA with a 5' overhang protruding end while Cas9 cleaves DNA with a blunt end. We have manipulated the murine calreticulin gene (calr) to introduce similar genetic defects as observed in human MPN using Cas9 and two kinds of CFP1 enzymes (AsCPF1 and LbCPF1). Efficiency of genetic alterations by the Cas9, AsCPF1 and LbCPF1 was compared. Furthermore, we used these enzymes to create Knock-In (KI) cells carrying an artificial exons in the last exon of calr .

<Methods> Two guide RNAs targeting the last exon of calr were designed for Cas9, AsCPF1 and LbCPF1. Vectors encoding the Cas9 or CPF1 nuclease with GFP/YFP as a marker were transfected into murine fibroblast cell line, NIH3T3, with the vectors encoding the guide RNAs. Transfected cells were sorted on a flow cytometer using the GFP/YFP as the marker. Single cell culture was performed in 96-well plates. Six, 11, 10 clones for Cas9, AsCPF1, LbCPF1 were isolated and nucleotide sequences of the last exon of calr were confirmed by direct sequencing. Expression of the transcripts from the altered alleles were also confirmed by reverse-transcription(RT) PCR and sequencing. Furthermore, we have created a KI construct in which the last exon of calr was divided into three artificial exons and the middle exon was flanked by LoxP sequences (floxed). The KI construct was tranfected into NIH3T3 with vectors encoding the Cas9 or CFP1 as well as vectors encoding the guide RNAs. Single clones were isolated and genetic status of the last exon of calr in each clone was examined by PCR/sequencing.

<Results> Nine of 11 clones for AsCPF1 (82%) and nine of 10 clones for LbCPF1 (90%) showed genetic abnormalities in the last exon of calr . Cas9 showed five manipulated clones of six clones (83%). Two, Three and Three clones were hemizygous abnormalities for Cas9, AsCPF1 and LbCPF1 respectively (33%, 27% and 30%). Three, four and four clones were compound-heterozygous abnormalities for Cas9, AsCPF1 and LbCPF1 respectively (50%, 36% and 40%). One clone for AsCPF1 and one clone for LbCPF1 were homozygous abnormalities. Majority of the genetic abnormalities were small deletion (1- 55bp). Three clones for Cas9 showed complete removal of the genome between the two target sites (168-170bp). One clone for Cas9 showed 163bp insertion in the target region. Altered messages transcribed from the manipulated alleles were confirmed by RT-PCR as well as sequencing in all the clones. As for the experiments to create the KI cells, more than 50 clones were isolated and one clone showed replacement of the last exon of calr by the KI construct. Transient expression of the Cre recommbinase by transfection efficiently deleted the floxed exon. However, we found that the KI cells expressed altered calr messages with complex sequence due to an unintentional insertion of the DNA fragment in the artificial exons.

<Discussion> With the Cas9, AsCPF1 and the LbCPF1 system, we successfully introduced genetic changes of calr in murine cell line. The efficacy of introducing genomic abnormalities into calr was similar among the three endonucleases, Cas9, AsCPF1, and LbCPF1. Small deletions in calr were efficiently introduced in the cell line. However, efficiency of KI was not sufficient. Optimization may be needed to create KI cell lines using the CRISPR/Cas9 system. Nevertheless, this technique can be applied for generation of murine models carrying the pathogenic calr allele or artificial conditional exons of calr .